Over ten million women suffer from urinary tract infection (UTI) annually. Most UTIs are caused by uropathogenic Escherichia coli (UPEC). Although antibiotics are typically effective, millions of women suffer from chronic recurrences and are often put on long-term suppressive and/or intravenous antibiotic therapy. Clearly, better therapeutic alternatives are critical for improving the lives of those suffering from UTI. Toward this end, studies of how UPEC cause recurrent UTI have revealed that pili assembled by the chaperone/usher pathway (CUP) play a critical role; abolishing the function of CUP pili renders UPEC noninfectious. CUP pili are comprised of multiple subunits with partial immunoglobulin folds. Previous work has shown that these partial folds are completed by adjacent subunits within the assembled pilus. Prior to assembly, a dedicated chaperone protein temporarily completes these folds. These exquisitely specific chaperone-subunit and subunit-subunit interactions are prime targets for treatment of UTI and other pili-related diseases. During infection, CUP pili mediate colonization, tissue invasion, and biofilm formation. Type 1 pili, for example, mediate the binding and invasion of UPEC into bladder epithelial cells, where they replicate rapidly in the cytoplasm. Simultaneously, the expression of type 1 pili facilitates the aggregation of the invaded organisms into a biofilm-like intracellular bacterial community (IBC) that protects the bacteria from host defenses and antibiotics. CUP pili are also important in biofilm formation on catheters and other surfaces in nosocomial settings. Individual E. coli can encode over ten CUP operons that likely are important in UTI or other infections, yet few have been studied. Therefore, an understanding of the contribution of CUP pili to biofilm formation and the regulation of their expression is also central to combating these infections. This proposal outlines a concerted research program to elucidate the complexity of fiber systems biology and its relation to disease. In vitro and in vivo models, genetics, imaging, and biochemical and biophysical techniques will be used to understand: fiber polymerization (aim 1); the role of CUP pili in biofilm formation (aim 2); and the regulation of expression of the multiple CUP operons in the E. coli genome (aim 3). These studies will increase the understanding of the fiber arsenal used by UPEC and other bacteria to cause problematic infections (such as UTI or catheter-associated infections). Knowledge of these pili is leading to the development of small molecules that block pilus assembly and/or function and thereby infection.